Forskolin induces circadian gene expression of rPer1, rPer2 and dbp in mammalian rat-1 fibroblasts

Mammalian culture cells have the potential for periodicity, since high concentrations of serum can elicit the circadian expression of clock genes in rat-1 fibroblasts. However, the mechanism by which serum affects circadian gene expression remains unclear. In the present study, we incubated rat-1 cells with forskolin and successfully induced the rhythmic expression of Per1, Per2 and dbp. In the initial step of the circadian gene expression, a marked transient induction of Per1 was observed accompanied with CREB phosphorylation. Thus the present study strongly suggests that CREB activation through the cAMP/PKA pathway is involved in the generation of circadian rhythm in rat-1 cells     

INI1 expression induces cell cycle arrest and markers of senescence in malignant rhabdoid tumor cells

The INI1 gene, which encodes a functionally uncharacterized protein component of the hSWI/SNF chromatin remodeling complex, is often mutated or deleted in malignant rhabdoid tumor (MRT). Two isoforms of INI1, that differ by the variable inclusion of nine amino acids, potentially are produced by differential RNA splicing. To determine the effect of the two INI1 isoforms on cell growth, INI1-devoid (MRT) and INI1-expressing cell lines were transfected separately with mammalian expression vectors or transduced with adenoviruses. Transfection of the short form of INI1 into either INI1-deficient or expressing cell lines resulted in complete suppression of cell growth in colony formation assays. The longer splice variant induced moderate to severe growth suppression of MRT cells, but had a far milder effect on non-MRT cells. Transduction of MRT cells with adenoviruses expressing either isoform of INI1 led to a dramatic change in morphology, growth suppression, and cell cycle arrest. Furthermore, senescence-associated proteins were up-regulated after transduction, while levels of proteins implicated in cell cycle progression were down-regulated. Adenoviral delivery of INI1 into a non-MRT cell line, however, had no demonstrable effect on any of these parameters. These results support the genetic evidence that INI1 is a tumor suppressor gene gone awry in MRT cells, and also suggest that delivery of the INI1 gene to MRT cells by adenoviruses may lead to a more effective treatment of this highly aggressive malignancy.     

Oncoprotein BMI‐1 induces the malignant transformation of HaCaT cells

BMI‐1 (B‐cell‐specific Moloney murine leukemia virus integration site 1), a novel oncogene, has attracted much attention in recent years for its involvement in the initiation of a variety of tumors. Recent evidence showed that BMI‐1 was highly expressed in neoplastic skin lesions. However, whether dysregulated BMI‐1 expression is causal for the transformation of skin cells remains unknown. In this study, we stably expressed BMI‐1 in a human keratinocyte cell line, HaCaT. The expression of wild‐type BMI‐1 induced the malignant transformation of HaCaT cells in vitro. More importantly, we found that expression of BMI‐1 promoted formation of squamous cell carcinomas in vivo. Furthermore, we showed that BMI‐1 expression led to the downregulation of tumore suppressors, such as p16INK4a and p14ARF, cell adhesion molecules, such as E‐Cadherin, and differentiation related factor, such as KRT6. Therefore, our findings demonstrated that dysregulated BMI‐1 could indeed lead to keratinocytes transformation and tumorigenesis, potentially through promoting cell cycle progression and increasing cell mobility. J. Cell. Biochem. 106: 16–24, 2009. © 2008 Wiley‐Liss, Inc.     

Constitutive expression of hyaluronan binding protein 1 (HABP1/p32/gC1qR) in normal fibroblast cells perturbs its growth characteristics and induces apoptosis

Hyaluronan binding protein 1 (HABP1) is a ubiquitously expressed multifunctional phospho-protein that interacts with a wide range of ligands and is implicated in cell signalling. Recently, we have reported that HABP1 is an endogenous substrate for MAP kinase and upon mitogenic stimulation it is translocated to the nucleus in a MAP kinase-dependent manner (Biochem. Biophys. Res. Commun. 291(4) (2002) 829-837). This prompted us to investigate the role of HABP1 in cell growth or otherwise in low MAP kinase background. We demonstrate that HABP1, when overexpressed in normal rat skin fibroblasts, remained in the cytosol, primarily concentrated around the nuclear periphery. However, HABP1 overexpressing cells showed extensive vacuolation and reduced growth rate, which was corrected by frequent medium replenishment. Further investigation revealed that HABP1 overexpressing cells undergo apoptosis, as detected by TUNEL assay, induction of Bax expression, and FACS analysis, and they failed to enter into the S-phase. Periodic medium supplementation prevented these cells from undergoing apoptotic death. We also demonstrate that upon induction of apoptosis in HeLa cells by cisplatin, HABP1 level is upregulated, indicating a correlation between HABP1 and cell death in a normal cellular environment.     

A short mitochondrial form of p19ARF induces autophagy and caspase-independent cell death

The tumor suppressor functions of p19(ARF) have been attributed to its ability to induce cell cycle arrest or apoptosis by activating p53 and regulating ribosome biogenesis. Here we describe another cellular function of p19(ARF), involving a short isoform (smARF, short mitochondrial ARF) that localizes to a Proteinase K-resistant compartment of the mitochondria. smARF is a product of internal initiation of translation at Met45, which lacks the nucleolar functional domains. The human p14(ARF) mRNA likewise produces a shorter isoform. smARF is maintained at low levels via proteasome-mediated degradation, but it increases in response to viral and cellular oncogenes. Ectopic expression of smARF reduces mitochondrial membrane potential (DeltaPsim) without causing cytochrome c release or caspase activation. The dissipation of DeltaPsim does not depend on p53 or Bcl-2 family members. smARF induces massive autophagy and caspase-independent cell death that can be partially rescued by knocking down ATG5 or Beclin-1, suggesting a different prodeath function for this short isoform.     

DeltaFosB,but not FosB,induces delayed apoptosis independent of cell proliferation in the Rat1a embryo cell line

The fates of Rat1a cells expressing FosB and DeltaFosB as fusion proteins (ER-FosB, ER-DeltaFosB) with the ligand binding domain of human estrogen receptor were examined. The binding of estrogen to the fusion proteins resulted in their nuclear translocation and triggered cell proliferation, and thereafter delayed cell death was observed only in cells expressing ER-DeltaFosB. The proliferation of Rat1a cells, but not cell death triggered by ER-DeltaFosB, was completely abolished by butyrolactone I, an inhibitor of cycline-dependent kinases, and was partly suppressed by antisense oligonucleotides against galectin-1, whose expression is induced after estrogen administration. The cell death was accompanied by the activation of caspase-3 and -9, the fragmentation of the nuclear genome and cytochrome c release from the mitochondria, and was suppressed by zDEVD-fmk and zLEHD-fmk but not zIETD-fmk. The cell death was not suppressed by exogenous His-PTD-Bcl-x(L) at all, suggesting involvement of a Bcl-x(L)-resistant pathway for cytochrome c release.     

Role of Pin1 in UVA-induced cell proliferation and malignant transformation in epidermal cells

Ultraviolet A (UVA) radiation (λ = 320–400 nm) is considered a major cause of human skin cancer. Pin1, a peptidyl prolyl isomerase, is overexpressed in most types of cancer tissues and plays an important role in cell proliferation and transformation. Here, we demonstrated that Pin1 expression was enhanced by low energy UVA (300–900 mJ/cm²) irradiation in both skin tissues of hairless mice and JB6 C141 epidermal cells. Exposure of epidermal cells to UVA radiation increased cell proliferation and cyclin D1 expression, and these changes were blocked by Pin1 inhibition. UVA irradiation also increased activator protein-1 (AP-1) minimal reporter activity and nuclear levels of c-Jun, but not c-Fos, in a Pin1-dependent manner. The increases in Pin1 expression and in AP-1 reporter activity in response to UVA were abolished by N-acetylcysteine (NAC) treatment. Finally, we found that pre-exposure of JB6 C141 cells to UVA potentiated EGF-inducible, anchorage-independent growth, and this effect was significantly suppressed by Pin1inhibition or by NAC.     

Role of Pin1 in UVA-induced cell proliferation and malignant transformation in epidermal cells

The hepatitis B virus X protein (HBx) has been implicated in the development of hepatocellular carcinoma (HCC) associated with chronic infection. As a multifunctional protein, HBx regulates numerous cellular pathways, including autophagy. Although autophagy has been shown to participate in viral DNA replication and envelopment, it remains unclear whether HBx-activated autophagy affects host cell death, which is relevant to both viral pathogenicity and the development of HCC. Here, we showed that enforced expression of HBx can inhibit starvation-induced cell death in hepatic (L02 and Chang) or hepatoma (HepG2 and BEL-7404) cell lines. Starvation-induced cell death was greatly increased in HBX-expressing cell lines treated either with the autophagy inhibitor 3-methyladenine (3-MA) or with an siRNA directed against an autophagy gene, beclin 1. In contrast, treatment of cells with the apoptosis inhibitor Z-Vad-fmk significantly reduced cell death. Our results demonstrate that HBx-mediated cell survival during starvation is dependent on autophagy. We then further investigated the mechanisms of cell death inhibition by HBx. We found that HBx inhibited the activation of caspase-3, an execution caspase, blocked the release of mitochondrial apoptogenic factors, such as cytochrome c and apoptosis-inducing factor (AIF), and inhibited the activation of caspase-9 during starvation. These results demonstrate that HBx reduces cell death through inhibition of mitochondrial apoptotic pathways. Moreover, increased cell viability was also observed in HepG2.2.15 cells that replicate HBV and in cells transfected with HBV genomic DNA. Our findings demonstrate that HBx promotes cell survival during nutrient deprivation through inhibition of apoptosis and activation of autophagy. This highlights an important potential role of autophagy in HBV-infected hepatocytes growing under nutrient-deficient conditions.     

Analysis of co-transfected expression vectors amplified by methotrexate selection in rat-1 cells.

In an earlier investigation of the influence of high level expression of p21H-ras, rat-1 cells were co-transfected with a selectable vector (pSV2Neo), an amplifiable vector (encoding dihydrofolate reductase; DHFR) and an H-ras expression vector. In this study we have analyzed the gene dose and expression levels of the three co-transfected plasmid vectors in cell lines that had been selected and isolated at different methotrexate concentrations. Growth of the cells in the absence of selection and Southern blot analyses indicate that the transfected vectors are stably co-integrated into the host genome. High expression levels from all three co-transfected vectors were evident at both the mRNA and protein levels, indicating that they are tightly linked in the host genome. The presence of a large amount of unspliced H-ras mRNA in cells expressing high levels of H-ras p21 indicates that processing of mRNA may be rate-limiting. Comparison of the gene dose and expression levels shows that the resistance of cells to increased methotrexate concentrations can occur by different mechanisms. It is concluded that co-transfection of individual plasmid vectors into rat-1 cells, followed by methotrexate selection, is an effective manner of achieving high level expression of proteins in cultured cells.     

Constitutive phosphorylation of eps8 in tumor cell lines: relevance to malignant transformation.

eps8, a recently identified tyrosine kinase substrate, has been shown to augment epidermal growth factor (EGF) responsiveness, implicating it in EGF receptor (EGFR)-mediated mitogenic signaling. We investigated the status of eps8 phosphorylation in normal and transformed cells and the role of eps8 in transformation. In NIH 3T3 cells overexpressing EGFR (NIH-EGFR), eps8 becomes rapidly phosphorylated upon EGF stimulation. At receptor-saturating doses of EGF, approximately 30% of the eps8 pool is tyrosine phosphorylated. Under physiological conditions of activation (i.e., at low receptor occupancy), corresponding to the 50% effective dose of EGF for mitogenesis, approximately 3 to 4% of the eps8 contains phosphotyrosine. In human tumor cell lines, we detected constitutive tyrosine phosphorylation of eps8, with a stoichiometry (approximately 5%) similar to that associated with potent mitogenic response in NIH-EGFR cells. Overexpression of eps8 was able to transform NIH 3T3 cells under limiting conditions of activation of the EGFR pathway. Concomitant tyrosine phosphorylation of eps8 and shc, but not of rasGAP, phospholipase C-gamma, and eps15, was frequently detected in tumor cells. This suggested that eps8 and shc might be part of a pathway which is preferentially selected in some tumors. Cooperation between these two transducers was further indicated by the finding of their in vivo association. This association was, at least in part, dependent on recognition of shc by the SH3 domain of eps8. Our results indicate that eps8 is physiologically part of the EGFR-activated signaling and that its alterations can contribute to the malignant phenotype.     

FosB gene products trigger cell proliferation and morphological alteration with an increased expression of a novel processed form of galectin-1 in the rat 3Y1 embryo cell line

In this study, we established rat 3Y1 embryo cell lines expressing FosB and DeltaFosB as fusion proteins (ER-FosB, ER-DeltaFosB) with the ligand-binding domain of human estrogen receptor (ER). The binding of estrogen to the fusion proteins resulted in their nuclear translocation. After estrogen administration, exponentially growing cells expressing ER-DeltaFosB, and to a lesser extent ER-FosB, underwent morphological alteration from the flat fibroblastic shape to an extended bipolar shape, and ceased proliferating. Such morphological alteration was also induced in quiescent cells expressing ER-DeltaFosB and ER-FosB after one round of cell division triggered by estrogen administration. The cells expressing ER-DeltaFosB changed shape frequently, and the content of F-actin in the cytoplasm detected by binding of Alexa 488-phalloidin significantly decreased after the morphological alteration. By two-dimensional gel electrophoresis analysis of cellular proteins from the cells expressing ER-DeltaFosB, we identified several proteins whose expression either increased or decreased after estrogen administration. Two of these proteins were identified from their amino acid sequences as novel processed form of galectin-1.     

Spontaneous, in vitro, malignant transformation of a basophil/mast cell line

It has recently become possible to grow basophil/mast cells in vitro for extended periods of time. Normally, these cultures remain fully dependent upon the presence of an adequate supply of growth factor(s) and the cells express several basophil/mast cell differentiated traits. We report here a case of spontaneous, in vitro, malignant transformation of such a basophil/mast cell line. The transformed cells no longer require the addition of growth factor(s) for continuous proliferation in vitro, and they have become highly tumorigenic in vivo. In contrast, when compared to their untransformed counterparts, they display the same set of differentiated traits, characteristic of immature basophil/mast cells. Thus, the data support the hypothesis that cell transformation results from a decreased sensitivity of precursor cells toward normal growth regulators but does not affect significantly the expression of differentiated functions.     

Ectopic expression of 10-formyltetrahydrofolate dehydrogenase in A549 cells induces G1 cell cycle arrest and apoptosis

We have recently shown that transient expression of 10-formyltetrahydrofolate dehydrogenase (FDH) strongly inhibits proliferation of several cancer cell lines and ultimately results in cell death. In the present studies using Tet-On system, we have generated a stable A549 lung carcinoma cell line capable of inducible FDH expression. Using this system, we were able to express FDH at different levels depending on concentration of the inducer, doxycycline, and we have observed that inhibition of proliferation depends on FDH intracellular levels. We have further shown that induction of FDH expression results in initiation of apoptosis beginning 24 h post-induction. Apoptotic cells revealed cleavage of poly-(ADP-ribose) polymerase and general caspase inhibitor zVAD-fmk protected cells against FDH-induced apoptosis. FDH-expressing cells showed accumulation of cells in G(0)-G(1) phase and a sharp decrease of cells in S phase. Accumulation of intracellular FDH was followed by accumulation of the tumor suppressor protein p53 and its downstream target p21. These results indicate that FDH antiproliferative effects on A549 cells include both G(1) cell cycle arrest and caspase-dependent apoptosis.